Although the initiation and maintenance of cellular differentiation are often invoked as a brake to tumorigenesis, the actual mechanisms by which differentiation could suppress cancer are largely unknown or hypothetical. In the case of pancreatic ductal adenocarcinoma (PDAC), the PIs of this application have identified a transcription factor network that constitutes a molecular link between differentiation and tumor suppression. At the heart of this network is the bHLH transcription factor PTF1A, a master regulator of gene expression in differentiated exocrine acinar cells. We and others have shown that although PDAC and its precancerous PanIN precursor lesions express markers of duct cells, they arise in mice from mutational activation of the KRAS oncogene in differentiated acinar cells rather than ducts. The first rate-limiting step of tumor initiation is acinar-ductal reprogramming, mediated by downregulation of the PTF1 network by KRAS and cooperating signals including EGFR. We hypothesize that PTF1 network activity determines the cellular and clinical effects of oncogenic KRAS: when PTF1 is active, acinar cells maintain normal differentiation even in the presence of mutant KRAS, and cancer is suppressed; by contrast, PTF1 downregulation unleashes oncogenic KRAS at the cellular level to produce invasive, lethal carcinoma. Using a combination of sophisticated mouse genetic models and novel, patient-derived human PDAC cell lines, with support by access to clinical samples, we will address both the mechanistic and translational aspects of this hypothesis in three aims: (1) determine if PTF1A is necessary and sufficient to suppress PanIN-to- PDAC progression, (2) determine if PTF1A suppresses PDAC initiation through inhibition of a tumor- promoting EGFR-SOX9 signaling axis, and (3) establish the role of the PTF1 network in normal human pancreas and human pancreatic cancer cells. These aims will be addressed with cutting-edge genetic, genomic and bioinformatic approaches developed in the laboratories of the two PIs, whose research programs provide complementary expertise in pancreatic cancer and pancreatic transcriptional regulation. Together, these studies will address the basic and clinical relevance of differentiation as a therapeutic target in pancreatic cancer.